Nanoprobe-metal chelator complexes

ABSTRACT

Provided herein are compounds that are able to bind metal ions (e.g., free metal ions or metal ions bound to low affinity ligands) in a sample or subject. Also provided herein are methods of using the compounds for chelating metal ions and for the treatment of diseases associated with abnormal levels of metal ions. Methods of preparing the compounds and pharmaceutical compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/484,722, filed Aug. 8, 2019, which is a § 371 national stageapplication of International Application No. PCT/US2017/039888, filed onJun. 29, 2017, which claims the benefit of U.S. Provisional ApplicationSer. No. 62/456,210, filed Feb. 8, 2017, the disclosure of which isincorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.R01-EB-011523 and R00-ES-017781, awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

TECHNICAL FIELD

Provided herein are compounds (e.g., nanochelator compounds) useful forbinding metal ions (e.g., free metal ions). Also provided are methods ofusing the compounds for chelating metal ions and treating diseasesassociated with abnormal levels of metal ions in a subject.

BACKGROUND

Iron is an essential metal, but high iron stores are toxic due toincreased oxidative stress produced by iron-catalyzed reactive oxygenspecies (ROS). Increased iron stores are also associated withwell-established risk factors of heart and liver failure, arthritis,dyslipidemia and diabetes, including obesity, metabolic syndrome andchronic inflammation (see e.g., Pietrangelo et al, Gastroenterology,2010, 139:393-408; Murphy et al, J. Card. Fail. 2010, 16:888-900;Fumeron et al, Diabetes Care, 2006, 29:2090-2094; Sun et al, The Journalof Clinical Endocrinology and Metabolism, 2008, 93:4690-4696; Vari etal, Diabetes Care, 2007, 30:1795-1801; and Casanova-Esteban et al,Metabolism: Clinical and Experimental, 60:830-834), particularly forindividuals with genetic susceptibility for primary and secondary ironoverload. Conversely, a reduction of iron stores by phlebotomy, ironchelation therapy, or iron-restricted diet has been shown to improveheart diseases, diabetes, and Alzheimer's disease (see e.g., Gulati etal, Cariol. Rev. 2014, 22:56-68; Sullivan et al, Recenti. Prog. Med.2007, 98:373-377; Bofill et al, Metabolism: Clinical and Experimental,1994, 43:614-620, Fernandex-Real, Diabetes, 2002, 51:1000-1004; and Fordet al, Diabetes Care, 1999, 22:1978-1983.

SUMMARY

The present application provides, inter alia, a compound of Formula I:A-B   Ior a pharmaceutically acceptable salt thereof, wherein:

A is a group comprising a zwitterion;

B is a biocompatible polymer substituted by one or more C groups and oneor more -D-E groups;

each C is independently selected from the group consisting of H and ananionic group;

each D is an independently selected linking group; and

each E is an independently selected metal chelating group.

In some embodiments, A comprises one or more cationic groups eachindependently selected from the group consisting of ammonium, C₁₋₆alkylammonium, di(C₁₋₆ alkyl)ammonium, tri(C₁₋₆ alkyl)ammonium, acationic 5-10 membered heteroaryl group, and a cationic 4-10 memberedheterocycloalkyl group, wherein the cationic 5-10 membered heteroarylgroup and cationic 4-10 membered heterocycloalkyl group are eachoptionally substituted by 1, 2, 3, or 4 independently selected C₁₋₆alkyl groups. In some embodiments, A comprises one or more cationicgroups which are each independently selected from the group consistingof tri(C₁₋₆ alkyl)ammonium and a cationic 5-10 membered heteroaryl groupwhich is optionally substituted by 1, 2, 3, or 4 independently selectedC₁₋₆ alkyl groups. In some embodiments, A comprises one or more cationicgroups which are each independently selected from the group consistingof trimethylammonium and N—(C₁₋₆ alkyl)indolium, wherein the N—(C₁₋₆alkyl)indolium is optionally substituted by 1 or 2 independentlyselected C₁₋₆ alkyl groups.

In some embodiments, A comprises one or more anionic groups eachindependently selected from the group consisting of oxide, carbonate,carboxylate, phosphate, sulfide, sulfinate, and sulfonate. In someembodiments, A comprises one or more anionic groups which are eachsulfonate.

In some embodiments, A is selected from the group consisting of formulasA-1 and A-2:

wherein:

indicates the bond between A and B;

X is selected from the group consisting of a bond, C, CH₂, NH, O and S;

each R^(A) is an independently selected anionic group;

-   -   each R^(C) is an independently selected cationic group; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group.

In some embodiments, each R^(C) is independently selected from the groupconsisting of ammonium, C₁₋₆ alkylammonium, di(C₁₋₆ alkyl)ammonium, andtri(C₁₋₆ alkyl)ammonium. In some embodiments, each R^(C) is anindependently selected tri(C₁₋₆ alkyl)ammonium group. In someembodiments, each R^(C) is trimethylammonium.

In some embodiments, each R^(A) is independently selected from the groupconsisting of oxide, carbonate, carboxylate, phosphate, sulfide,sulfinate, and sulfonate.

In some embodiments, each R^(A) is sulfonate.

In some embodiments, L¹ is propylene.

In some embodiments, L² is propylene.

In some embodiments, L³ is ethylene.

In some embodiments, X is a bond. In some embodiments, X is CH₂.

In some embodiments, A is:

In some embodiments, B is selected from the group consisting of abiocompatible polypeptide and a biocompatible polyester, each of whichis substituted by one or more C groups and one or more -D-E groups. Insome embodiments, B is selected from the group consisting of polylysine,polylactic acid, poly(lactic-co-glycolic acid), polyaspartic acid,polyglutamic acid, and polyglutamic acid-poly(ethylene glycol)copolymer, each of which is substituted by one or more C groups and oneor more -D-E groups. In some embodiments, B is polylysine substituted byone or more C groups and one or more -D-E groups. In some embodiments,the polylysine is ε-poly-L-lysine substituted by one or more C groupsand one or more -D-E groups.

In some embodiments, B is:

wherein:

indicates the bond between B and A; and

n is an integer from 5 to 30; and

m is an integer from 1 to 10.

In some embodiments, B has a hydrodynamic radius of from about 1 nm toabout 10 nm.

In some embodiments, each C is independently selected from the groupconsisting of hydrogen and an anionic group comprising one or morealkylene groups, one or more carbonyl groups, or one or more carboxylgroups, or any combination thereof.

In some embodiments, C is an anionic group of the following formula:

wherein:

indicates the bond between C and B; and

p is an integer from 1 to 10.

In some embodiments, p is an integer from 1 to 5.

In some embodiments, D is a linking group comprising one or morealkylene groups, one or more carbonyl groups, or one or more carboxylgroups, or any combination thereof.

In some embodiments, D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 10.

In some embodiments, q is an integer from 1 to 5.

In some embodiments, E is selected from the group consisting of an ironchelating group, a lead chelating group, a copper chelating group, anarsenic chelating group, a mercury chelating group, and a manganesechelating group. In some embodiments, E is an iron chelating group. Insome embodiments, E is selected from the group consisting ofdimercaptosuccinic acid, dimercaprol, ethylenediaminetetraacetic acid,p-aminosalicyclic acid, D-penicillamine, deferoxamine, deferiprone, anddeferasirox. In some embodiments, E is deferoxamine.

In some embodiments, B is polylysine and E is an iron chelating group,wherein the polylysine is substituted by one or more C groups and one ormore -D-E groups. In some embodiments, the polylysine is ε-poly-L-lysine(i.e., EPL) substituted by one or more C groups and one or more -D-Egroups. In some embodiments, B is ε-poly-L-lysine and E is deferoxamine(i.e., DFO), wherein the ε-poly-L-lysine is substituted by one or more Cgroups and one or more -D-E groups.

In some embodiments, the molar ratio (i.e., stoichiometry) of the metalchelating agent E (e.g., an iron chelating agent) to the biocompatiblepolymer B in the compounds of Formula I is from about 10:1 to about 1:1,for example, about 10:1 to about 2:1, about 10:1 to about 4:1, about10:1 to about 6:1, about 10:1 to about 8:1, about 8:1 to about 1:1,about 8:1 to about 2:1, about 8:1 to about 4:1, about 8:1 to about 6:1,about 6:1 to about 1:1, about 6:1 to about 2:1, about 6:1 to about 4:1,about 4:1 to about 1:1, about 4:1 to about 2:1, or about 2:1 to about1:1.

In some embodiments, the molar ratio (i.e., stoichiometry) of the metalchelating agent E (e.g., an iron chelating agent) to the biocompatiblepolymer B in the compounds of Formula I is about 2:1, about 4:1, about6:1, or about 8:1.

In some embodiments:

A is selected from the group consisting of formulas A-1 and A-2:

wherein:

indicates the bond between A and B;

each R^(A) is an independently selected anionic group;

each R^(C) is an independently selected cationic group; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group;

B is selected from the group consisting of a biocompatible polypeptideand a biocompatible polyester, each of which is substituted by one ormore C groups and one or more -D-E groups;

C is an anionic group of the following formula:

wherein:

indicates the bond between C and B;

p is an integer from 1 to 10;

D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 10; and

E is a metal chelating group.

In some embodiments:

A is a group of the following formula:

wherein:

indicates the bond between A and B;

each R^(A) is independently selected from the group consisting of oxide,carbonate, carboxylate, phosphate, sulfide, sulfinate, and sulfonate;

each R^(C) is independently selected from the group consisting ofammonium, C₁₋₆ alkylammonium, di(C₁₋₆ alkyl)ammonium, and tri(C₁₋₆alkyl)ammonium; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group;

B is selected from the group consisting of polylysine, polylactic acid,and poly(lactic-co-glycolic acid), polyaspartic acid, polyglutamic acid,and polyglutamic acid-poly(ethylene glycol) copolymer, each of which aresubstituted by one or more C groups and one or more -D-E groups;

C is an anionic group of the following formula:

wherein:

indicates the bond between C and B;

p is an integer from 1 to 10;

D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 10; and

E is a metal chelating group.

In some embodiments:

A is a group of the following formula:

wherein:

indicates the bond between A and B;

each R^(A) is sulfonate;

each R^(C) is tri(C₁₋₆ alkyl)ammonium; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group;

B is polylysine which is substituted by one or more C groups and one ormore -D-E groups;

C is an anionic group of the following formula:

wherein:

indicates the bond between C and B;

p is an integer from 1 to 5;

D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 5; and

E is a metal chelating group.

In some embodiments, E is selected from the group consisting of an ironchelating group, a lead chelating group, and a copper chelating group.In some embodiments, E is an iron chelating group. In some embodiments,E is selected from the group consisting of dimercaptosuccinic acid,dimercaprol, ethylenediaminetetraacetic acid, P-aminosalicyclic acid,D-penicillamine, deferoxamine, deferiprone, and deferasirox. In someembodiments, E is deferoxamine.

The present application further provides a pharmaceutical compositioncomprising the compound provided herein (e.g., a compound of Formula I),or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

The present application further provides a method of chelating a metalion in a cell or tissue sample, comprising contacting the cell sample ortissue sample with a compound provided herein (e.g., a compound ofFormula I), or a pharmaceutically acceptable salt thereof.

The present application further provides a method of reducing the amountof free metal ions in a cell or tissue sample, comprising contacting thecell or tissue sample with a compound provided herein (e.g., a compoundof Formula I), or a pharmaceutically acceptable salt thereof.

The present application further provides a method of chelating metalions in a subject, comprising administering to the subject atherapeutically effective amount of a compound provided herein (e.g., acompound of Formula I), or a pharmaceutically acceptable salt thereof.

The present application further provides a method of reducing the amountof free metal ions in a subject, comprising administering to the subjecta compound provided herein (e.g., a compound of Formula I), or apharmaceutically acceptable salt thereof.

The present application further provides a method of reducing the amountof free metal ions in a subject, comprising:

i) diagnosing the subject as having an abnormal level of free metalions; and

ii) administering to the subject a therapeutically effective amount of acompound provided herein (e.g., a compound of Formula I), or apharmaceutically acceptable salt thereof.

The present application further provides a method of reducing the amountof free metal ions in a subject, comprising administering to a subjectdetermined to have an abnormal level of free metal ions atherapeutically effective amount of a compound provided herein (e.g., acompound of Formula I), or a pharmaceutically acceptable salt thereof.

The present application further provides a method of treating a diseaseassociated with an abnormal amount of free metal ions in a subject,comprising administering to a subject determined to have an abnormallevel of free metal ions a compound provided herein (e.g., a compound ofFormula I), or a pharmaceutically acceptable salt thereof.

In some embodiments, the disease is associated with an abnormal amountof iron ions, an abnormal amount of lead ions, or an abnormal amount ofcopper ions in the subject, or any combination thereof. In someembodiments, the disease is associated with an abnormal amount of ironions in the subject.

In some embodiments, the disease is selected from the group consistingof transfusion hemosiderosis, hemochromatosis, Wilson's disease, copperpoisoning, and heavy metal poisoning.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show the ultra-violet (UV) absorption spectra ofnanochelators ZW-EPL⁻ and DFO-ZW-EPL⁻.

FIGS. 1C-1E show the optical properties of nanochelator DFO-ZW-EPL⁻(FIG. 1C) and the HPLC spectra of DFO-ZW-EPL⁻ (FIGS. 1D-1E).

FIGS. 1F-1G show absorbance of the DFO-ZW-EPL⁻ nanochelators measured ina UV assay (FIG. 1F) and an Fe assay (FIG. 1G).

FIGS. 2A-2C shows that the DFO-ZW-EPL⁻ nanochelator decreases ironconcentration in serum from iron overloaded animals. FIG. 2A shows ironbinding capacity in TBS; FIG. 2B shows iron binding capacity in serumfrom mice with iron overload hemochromatosis; and FIG. 2C shows ironbinding capacity in serum from rats with iron overload hemoglobinopathy.

FIGS. 3A-3B show the biodistribution and clearance of ZW-EPL⁻ andDFO-ZW-EPL⁻ in CD-1 mice. Abbreviations: Bl, bladder; Du, duodenum; He,Heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa,pancreas; Sp, spleen. Scale bars=1 cm.

FIGS. 4A-4C show clearance (FIGS. 4A-4B) of ZW-EPL⁻ and DFO-ZW-EPL⁻ overtime and biodistribution of the compounds in representative organs (FIG.4C). Abbreviations: Bl, bladder; Du, duodenum; He, Heart; In, intestine;Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen.

FIGS. 5A-5B shows in vivo iron chelation effects of DFO-ZW-EPL⁻ indietary iron overloaded rats. Serum iron and liver non-heme iron levelswere quantified by non-heme iron analysis.

FIGS. 6A-6B shows in vivo iron chelation effects of DFO-ZW-EPL⁻ in HFEmutant mice with genetic iron overload.

FIGS. 6C-6D show in vivo effects on serum iron levels in HFE mutant miceadministered DFO-ZW-EPL⁻ nanochelators.

FIGS. 7A-7B show that DFO-ZW-EPL⁻ may correct cardiac hypertrophy inHFE-knockout mice.

FIGS. 8A-8G show that DFO-ZW-EPL⁻ may correct dyslipidemia in Belgraderats.

FIG. 9 shows in vivo iron recovery in urine in CD-1 mice treated withiron-dextran and DFO-ZW-EPL⁻ nanochelators.

FIG. 10 shows representative optical and NIR fluorescence images ofkidneys from CD-1 mice treated with iron-dextran and DFO-ZW-EPL⁻nanochelators in single dose and multi-dose experiments.

DETAILED DESCRIPTION

Because there is no recognized active pathway of iron excretion, adisposal of extra iron from the body is the primary therapeutic goal oftreating patients with iron overload. The chelation therapy has beenused to improve disease conditions in patients with iron overload,particularly transfusion-associated iron loading. However, ironchelators have serious adverse effects. While there are threeFDA-approved iron chelators, the use of these chelators is limitedbecause of non-specific distribution in non-target tissues, which mayresult in toxicities including hypotension, tachycardia,agranulocytosis, neutropenia, central nervous system (CNS) andocular/auditory toxicity, musculoskeletal joint pains, gastrointestinaldisturbances, and even death. There is therefore a need to establish newtherapeutic strategies using safer chelators.

Accordingly, the present application provides compound (e.g., nanoprobesor “NPs”) that covalently bind metal chelators (e.g., “nanochelators”)and thereby limit drug distribution into non-target tissues, whileefficiently capturing plasma metal ions (e.g., iron ions) and beingcleared, for example, via urine.

Compounds

The present application provides a compound of Formula I:A-B   Ior a pharmaceutically acceptable salt thereof, wherein:

A is a group comprising a zwitterion;

B is a biocompatible polymer substituted by one or more C groups and oneor more -D-E groups;

each C is independently selected from the group consisting of H and ananionic group;

each D is an independently selected linking group; and

each E is an independently selected metal chelating group.

In some embodiments, A comprises one or more cationic groups eachindependently selected from the group consisting of ammonium, C₁₋₆alkylammonium, di(C₁₋₆ alkyl)ammonium, tri(C₁₋₆ alkyl)ammonium, acationic 5-10 membered heteroaryl group, and a cationic 4-10 memberedheterocycloalkyl group, wherein the cationic 5-10 membered heteroarylgroup and cationic 4-10 membered heterocycloalkyl group are eachoptionally substituted by 1, 2, 3, or 4 independently selected C₁₋₆alkyl groups. In some embodiments, A comprises one or more cationicgroups which are each independently selected from the group consistingof tri(C₁₋₆ alkyl)ammonium and a cationic 5-10 membered heteroaryl groupwhich is optionally substituted by 1, 2, 3, or 4 independently selectedC₁₋₆ alkyl groups. In some embodiments, A comprises one or more cationicgroups which are each independently selected from the group consistingof trimethylammonium and N—(C₁₋₆ alkyl)indolium, wherein the N—(C₁₋₆alkyl)indolium is optionally substituted by 1 or 2 independentlyselected C₁₋₆ alkyl groups.

In some embodiments, A comprises 1, 2, 3, or 4 cationic groups eachindependently selected from the group consisting of ammonium, C₁₋₆alkylammonium, di(C₁₋₆ alkyl)ammonium, tri(C₁₋₆ alkyl)ammonium, acationic 5-10 membered heteroaryl group, and a cationic 4-10 memberedheterocycloalkyl group, wherein the cationic 5-10 membered heteroarylgroup and cationic 4-10 membered heterocycloalkyl group are eachoptionally substituted by 1, 2, 3, or 4 independently selected C₁₋₆alkyl groups.

In some embodiments, A comprises 1, 2, 3, or 4 cationic groups which areeach independently selected from the group consisting of tri(C₁₋₆alkyl)ammonium and a cationic 5-10 membered heteroaryl group which isoptionally substituted by 1, 2, 3, or 4 independently selected C₁₋₆alkyl groups.

In some embodiments, A comprises 1, 2, 3, or 4 cationic groups which areeach independently selected from the group consisting oftrimethylammonium and N—(C₁₋₆ alkyl)indolium, wherein the N—(C₁₋₆alkyl)indolium is optionally substituted by 1 or 2 independentlyselected C₁₋₆ alkyl groups.

In some embodiments, A comprises one or more anionic groups eachindependently selected from the group consisting of oxide, carbonate,carboxylate, phosphate, sulfide, sulfinate, and sulfonate. In someembodiments, A comprises one or more anionic groups which are eachsulfonate.

In some embodiments, A comprises 1, 2, 3, or 4 anionic groups eachindependently selected from the group consisting of oxide, carbonate,carboxylate, phosphate, sulfide, sulfinate, and sulfonate. In someembodiments, A comprises 1 or 2 anionic groups each independentlyselected from the group consisting of oxide, carbonate, carboxylate,phosphate, sulfide, sulfinate, and sulfonate.

In some embodiments, A comprises 1, 2, 3, or 4 anionic groups which areeach sulfonate. In some embodiments, A comprises 1 or 2 anionic groupswhich are each sulfonate.

In some embodiments, A is selected from the group consisting of formulasA-1, A-2, A-3, A-4, A-5, and A-6:

wherein:

indicates the bond between A and B;

X is selected from the group consisting of a bond, CH₂, NH, —NH—C₁₋₆alkylene-, O, and S;

each R^(A) is an independently selected anionic group;

each R^(C) is an independently selected cationic group; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group.

In some embodiments, A is selected from the group consisting of formulasA-1 and A-2:

wherein:

indicates the bond between A and B;

X is selected from the group consisting of a bond, C, CH₂, NH, O and S;

each R^(A) is an independently selected anionic group;

each R^(C) is an independently selected cationic group; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group

In some embodiments, each R^(C) is independently selected from the groupconsisting of ammonium, C₁₋₆ alkylammonium, di(C₁₋₆ alkyl)ammonium, andtri(C₁₋₆ alkyl)ammonium. In some embodiments, each R^(C) is anindependently selected tri(C₁₋₆ alkyl)ammonium group. In someembodiments, each R^(C) is trimethylammonium.

In some embodiments, each R^(A) is independently selected from the groupconsisting of oxide, carbonate, carboxylate, phosphate, sulfide,sulfinate, and sulfonate. In some embodiments, each R^(A) is sulfonate.

In some embodiments, L¹ is a C₁₋₃ alkylene group. In some embodiments,L¹ is propylene.

In some embodiments, L² is a C₁₋₃ alkylene group. In some embodiments,L² is propylene.

In some embodiments, L¹ and L² are each an independently selected C₁₋₃alkylene group. In some embodiments, L¹ and L² are the same. In someembodiments, L¹ and L² are different. In some embodiments, L¹ and L² areeach propylene.

In some embodiments, L³ is a C₁₋₃ alkylene group. In some embodiments,L³ is ethylene.

In some embodiments, X is a bond. In some embodiments, X is selectedfrom the group consisting of C, CH₂, NH, —NH—C₁₋₆ alkylene-, O, and S.In some embodiments, X is CH₂. In some embodiments, X is selected fromthe group consisting of NH, —NH—C₁₋₆ alkylene-, O, and S.

In some embodiments, A is selected from the group consisting of:

In some embodiments, A is:

In some embodiments, B is selected from the group consisting of abiocompatible polypeptide and a biocompatible polyester, each of whichare substituted by one or more C groups and one or more -D-E groups.

In some embodiments, B is selected from the group consisting ofpolylysine, polylactic acid, poly(lactic-co-glycolic acid), polyasparticacid, polyglutamic acid, and polyglutamic acid-poly(ethylene glycol)copolymer, each of which are substituted by one or more C groups and oneor more -D-E groups. In some embodiments, B is polylysine substituted byone or more C groups and one or more -D-E groups. In some embodiments,the polylysine is ε-poly-L-lysine substituted by one or more C groupsand one or more -D-E groups.

In some embodiments, B is:

wherein:

indicates the bond between B and A; and

n is an integer from 2 to 50; and

m is an integer from 1 to 20.

In some embodiments, n is an integer from 2 to 30, for example, 2 to 30,2 to 20, 2 to 10, 2 to 5, 5 to 30, 5 to 20, 5 to 10, 10 to 30, 10 to 20,or 20 to 30.

In some embodiments, m is an integer from 1 to 10, for example, 1 to 10,1 to 5, 1 to 3, 3 to 10, 3 to 5, or 5 to 10.

In some embodiments, n is an integer from 5 to 30 and m is an integerfrom 1 to 10.

In some embodiments, the hydrodynamic radius of B is from about 1 nm toabout 10 nm, for example, about 1 nm to about 10 nm, about 1 nm to about8 nm, about 1 nm to about 6 nm, about 1 nm to about 4 nm, about 1 nm toabout 2 nm, about 2 nm to about 10 nm, about 2 nm to about 8 nm, about 2nm to about 6 nm, about 2 nm to about 4 nm, about 4 nm to about 10 nm,about 4 nm to about 8 nm, about 4 nm to about 6 nm, about 6 nm to about10 nm, about 6 nm to about 8 nm, or about 8 nm to about 10 nm.

In some embodiments, each C is independently selected from the groupconsisting of hydrogen and an anionic group comprising one or morealkylene groups, one or more carbonyl groups, and one or more carboxylgroups.

In some embodiments, C is an anionic group of the following formula:

wherein:

indicates the bond between C and B;

p is an integer from 1 to 10.

In some embodiments,

In some embodiments, p is an integer from 1 to 5, for example, 1 to 5, 1to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, or 4 to 5.

In some embodiments, D is a linking group comprising one or morealkylene groups, one or more carbonyl groups, and one or more carboxylgroups.

In some embodiments, D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 10.

In some embodiments, q is an integer from 1 to 5, for example, 1 to 5, 1to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, or 4 to 5.

In some embodiments, A is a group of formula A-1 or A-2:

wherein:

indicates the bond between A and B;

each R^(A) is an independently selected anionic group;

each R^(C) is an independently selected cationic group; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group;

B is selected from the group consisting of a biocompatible polypeptideand a biocompatible polyester, each of which are substituted by one ormore C groups and one or more -D-E groups;

C is an anionic group of the following formula:

wherein:

indicates the bond between C and B;

p is an integer from 1 to 10;

D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 10; and

E is a metal chelating group.

In some embodiments:

A is a group of the following formula:

wherein:

indicates the bond between A and B;

each R^(A) is independently selected from the group consisting of oxide,carbonate, carboxylate, phosphate, sulfide, sulfinate, and sulfonate;

each R^(C) is independently selected from the group consisting ofammonium, C₁₋₆ alkylammonium, di(C₁₋₆ alkyl)ammonium, and tri(C₁₋₆alkyl)ammonium; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group;

B is selected from the group consisting of polylysine, polylactic acid,and poly(lactic-co-glycolic acid), polyaspartic acid, polyglutamic acid,and polyglutamic acid-poly(ethylene glycol) copolymer, each of which aresubstituted by one or more C groups and one or more -D-E groups;

C is an anionic group of the following formula:

wherein:

indicates the bond between C and B;

p is an integer from 1 to 10;

D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 10; and

E is a metal chelating group.

In some embodiments:

A is a group of the following formula:

wherein:

indicates the bond between A and B;

each R^(A) is sulfonate;

each R^(C) is tri(C₁₋₆ alkyl)ammonium; and

L¹, L², and L³ are each an independently selected C₁₋₆ alkylene group; Bis polylysine which is substituted by one or more C groups and one ormore -D-E groups;

C is an anionic group of the following formula:

wherein:

indicates the bond between C and B;

p is an integer from 1 to 5;

D is a linking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and

q is an integer from 1 to 5; and

E is a metal chelating group.

In some embodiments, E is selected from the group consisting of an ironchelating group, a lead chelating group, and a copper chelating group.In some embodiments, E is an iron chelating group.

In some embodiments, E is selected from the group consisting of an ironchelating group, a lead chelating group, a copper chelating group, anarsenic chelating group, a mercury chelating group, a manganesechelating group, a cadmium chelating group, a nickel chelating group, achromium chelating group, a gold chelating group, and an antimonychelating group.

Example metal chelating groups include, but are not limited to,deferoxamine, deferasirox and deferiprone (e.g., for chelating iron),D-penicillamine (e.g., for chelating copper), dimercaprol, (e.g., forchelating arsenic, mercury, lead, cadmium, nickel, chromium, gold,and/or antimony), dimercaptosuccinic acid (DMSA) (e.g., for chelatingarsenic mercury, and/or lead), Calcium disodium EDTA (e.g., forchelating mercury and/or lead), and p-aminosalicyclic acid (e.g., forchelating manganese).

In some embodiments, E is selected from the group consisting of an ironchelating group, a lead chelating group, a copper chelating group, anarsenic chelating group, a mercury chelating group, and a manganesechelating group. In some embodiments, E is an iron chelating group.

In some embodiments, E is selected from the group consisting ofdimercaptosuccinic acid, dimercaprol, ethylenediaminetetraacetic acid,p-aminosalicyclic acid, D-penicillamine, deferoxamine, deferiprone, anddeferasirox. In some embodiments, E is deferoxamine.

In some embodiments, the compound of Formula I is:

or a pharmaceutically acceptable salt thereof, wherein groups n, m,ZW800, and DFO are defined according to the definitions provided herein.Synthesis

The present application further provides methods of preparing thecompounds provided herein and salts thereof. For example, the compoundsprovided herein (e.g., compounds of Formula I) and salts thereof, can beprepared according to the procedure shown below in Scheme 1.

It will be appreciated by one skilled in the art that the processesdescribed are not the exclusive means by which compounds provided hereinmay be synthesized and that a broad repertoire of synthetic organicreactions is available to be potentially employed in synthesizingcompounds provided herein. The person skilled in the art knows how toselect and implement appropriate synthetic routes. Suitable syntheticmethods of starting materials, intermediates and products may beidentified by reference to the literature, including reference sourcessuch as: Advances in Heterocyclic Chemistry, Vols. 1-107 (Elsevier,1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal ofHeterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science ofSynthesis, Vols. 1-48 (2001-2010) and Knowledge Updates KU2010/1-4;2011/1-4; 2012/1-2 (Thieme, 2001-2012); Katritzky, et al. (Ed.)Comprehensive Organic Functional Group Transformations, (Pergamon Press,1996); Katritzky et al. (Ed.); Comprehensive Organic Functional GroupTransformations II (Elsevier, 2^(nd) Edition, 2004); Katritzky et al.(Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984);Katritzky et al., Comprehensive Heterocyclic Chemistry II, (PergamonPress, 1996); Smith et al., March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 6^(th) Ed. (Wiley, 2007); Trost etal. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

Preparation of compounds described herein can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., Wiley &Sons, Inc., New York (1999).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC), liquid chromatography-mass spectroscopy(LCMS), or thin layer chromatography (TLC). Compounds can be purified bythose skilled in the art by a variety of methods, including highperformance liquid chromatography (HPLC) and normal phase silicachromatography.

At various places in the present specification, divalent linkingsubstituents are described. It is specifically intended that eachdivalent linking substituent include both the forward and backward formsof the linking substituent. For example, —NR(CR′R″)_(n)-includes both—NR(CR′R″)_(n)— and —(CR′R″)_(n)NR—. Where the structure clearlyrequires a linking group, the Markush variables listed for that groupare understood to be linking groups.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is to beunderstood that substitution at a given atom is limited by valency.

Throughout the definitions, the term “C_(n-m)” indicates a range whichincludes the endpoints, wherein n and m are integers and indicate thenumber of carbons. Examples include C₁₋₄, C₁₋₆, and the like.

As used herein, the term “C_(n-m) alkyl” refers to a saturatedhydrocarbon group that may be straight-chain or branched, having n to mcarbons. Examples of alkyl moieties include, but are not limited to,chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,tert-butyl, isobutyl, sec-butyl; higher homologs such as2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl,and the like. In some embodiments, the alkyl group contains from 1 to 6carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1to 2 carbon atoms.

As used herein, the term “C_(n-m) alkylene”, employed alone or incombination with other terms, refers to a divalent alkyl linking grouphaving n to m carbons. Examples of alkylene groups include, but are notlimited to, ethan-1,1-diyl, ethan-1,2-diyl, propan-1,1,-diyl,propan-1,3-diyl, propan-1,2-diyl, butan-1,4-diyl, butan-1,3-diyl,butan-1,2-diyl, 2-methyl-propan-1,3-diyl, and the like. In someembodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to6, 1 to 4, or 1 to 2 carbon atoms.

As used herein, the term “ammonium” refers to a group of formula —NH₃ ⁺.

As used herein, the term “C_(n-m) alkylammonium” refers to a group offormula [NH₂(C_(n-m) alkyl)]⁺, wherein the C_(n-m) alkyl refers to asaturated hydrocarbon group that may be straight-chain or branched,having n to m carbons, as defined herein.

As used herein, the term “di(C_(n-m) alkyl)ammonium” refers to a groupof formula —[NH(C_(n-m) alkyl)₂]⁺, wherein the each C_(n-m) alkyl refersto an independently selected saturated hydrocarbon group that may bestraight-chain or branched, having n to m carbons, as defined herein.

As used herein, the term “tri(C_(n-m) alkyl)ammonium” refers to a groupof formula —[N(C_(n-m) alkyl)₃]⁺, wherein the each C_(n-m) alkyl refersto an independently selected saturated hydrocarbon group that may bestraight-chain or branched, having n to m carbons, as defined herein.

As used herein, the term “carboxylate” refers to a group of formula“—COO⁻”.

As used herein, the term “carbonate” refers to a group of formula “—CO₃²⁻”. As used herein, the term “heteroaryl” refers to a monocyclicaromatic heterocycle having at least one heteroatom ring member selectedfrom sulfur, oxygen, and nitrogen. In some embodiments, the heteroarylring has 1, 2, 3, or 4 heteroatom ring members independently selectedfrom nitrogen, sulfur and oxygen. In some embodiments, the heteroarylring has 1, 2, 3, or 4 heteroatom ring members independently selectedfrom nitrogen and sulfur. In some embodiments, any ring-forming N in aheteroaryl moiety can form an N-oxide. In some embodiments, theheteroaryl has 5-6 ring atoms and 1, 2, 3, or 4 heteroatom ring membersindependently selected from nitrogen, sulfur and oxygen. In someembodiments, the heteroaryl has 5-6 ring atoms and 1, 2, 3, or 4heteroatom ring members independently selected from nitrogen and sulfur.In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2heteroatom ring members independently selected from nitrogen and sulfur.Exemplary five-membered ring heteroaryl groups include, but are notlimited to, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl,pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl,1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. Exemplary six-membered ringheteroaryl groups include, but are not limited to, pyridyl, pyrazinyl,pyrimidinyl, triazinyl and pyridazinyl.

It is understood that a cationic heteroaryl group refers to a heteroarylgroup as defined herein having one or more positive charges. Exemplarycationic heteroaryl groups include, but are not limited to, pyridium,pyrazinium, pyrimidinium, triazinium, and indolium.

As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic orpolycyclic heterocycles having one or more ring-forming heteroatomsselected from 0, N, or S. Included in heterocycloalkyl are monocyclic4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkylgroups can also include spirocycles. Example heterocycloalkyl groupsinclude pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl,tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino,piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, andthe like. Ring-forming carbon atoms and heteroatoms of aheterocycloalkyl group can be optionally substituted by oxo or sulfido(e.g., C(O), S(O), C(S), or S(O)2, etc.). The heterocycloalkyl group canbe attached through a ring-forming carbon atom or a ring-formingheteroatom. In some embodiments, the heterocycloalkyl group contains 0to 3 double bonds. In some embodiments, the heterocycloalkyl groupcontains 0 to 2 double bonds. Also included in the definition ofheterocycloalkyl are moieties that have one or more aromatic rings fused(i.e., having a bond in common with) to the cycloalkyl ring, forexample, benzo or thienyl derivatives of piperidine, morpholine,azepine, etc. A heterocycloalkyl group containing a fused aromatic ringcan be attached through any ring-forming atom including a ring-formingatom of the fused aromatic ring. In some embodiments, theheterocycloalkyl has 4-10, 4-7 or 4-6 ring atoms with 1 or 2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur and having oneor more oxidized ring members.

It is understood that a cationic heterocycloalkyl group refers to aheterocycloalkyl group as defined herein having one or more positivecharges. Exemplary cationic heterocycloalkyl groups include, but are notlimited to, oxetanium, azetidinium, morpholinium, and thiomorpholinium.

At certain places, the definitions or embodiments refer to specificrings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwiseindicated, these rings can be attached to any ring member provided thatthe valency of the atom is not exceeded. For example, an azetidine ringmay be attached at any position of the ring, whereas a pyridin-3-yl ringis attached at the 3-position.

As used herein, the term “oxide” refers to a group of formula “—O⁻ ”.

As used herein, the term “phosphate” refers to a group of formula “—PO₄³⁻”.

As used herein, the term “sulfide” refers to a group of formula “—S⁻”.

As used herein, the term “sulfinate” refers to a group of formula “—SO₂⁻”.

As used herein, the term “sulfonate” refers to a group of formula “—SO₃⁻”.

As used herein, the term “zwitterion” refers to a group comprising oneor more positively charged groups (e.g., ammonium, C₁₋₆ alkylammonium,di(C₁₋₆ alkyl)ammonium, tri(C₁₋₆ alkyl)ammonium, and the like) and oneor more negatively charged groups (e.g., sulfinate, sulfonate,phosphate, oxide, and the like).

As used herein, the term “metal ions” refers to free metal ions or metalions bound to low affinity ligands (e.g., citrate), or a combinationthereof, in a sample (e.g., a cell sample or tissue sample) or asubject.

The term “compound” as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted. Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified.

Compounds provided herein also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone -enol pairs, amide -imidic acidpairs, lactam -lactim pairs, enamine -imine pairs, and annular formswhere a proton can occupy two or more positions of a heterocyclicsystem, for example, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.hydrates and solvates) or can be isolated.

In some embodiments, preparation of compounds can involve the additionof acids or bases to affect, for example, catalysis of a desiredreaction or formation of salt forms such as acid addition salts.

Example acids can be inorganic or organic acids and include, but are notlimited to, strong and weak acids. Some example acids includehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid,benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weakacids include, but are not limited to acetic acid, propionic acid,butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoicacid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

Example bases include lithium hydroxide, sodium hydroxide, potassiumhydroxide, lithium carbonate, sodium carbonate, potassium carbonate, andsodium bicarbonate. Some example strong bases include, but are notlimited to, hydroxide, alkoxides, metal amides, metal hydrides, metaldialkylamides and arylamines, wherein; alkoxides include lithium, sodiumand potassium salts of methyl, ethyl and t-butyl oxides; metal amidesinclude sodium amide, potassium amide and lithium amide; metal hydridesinclude sodium hydride, potassium hydride and lithium hydride; and metaldialkylamides include lithium, sodium, and potassium salts of methyl,ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl andcyclohexyl substituted amides.

In some embodiments, the compounds and salts provided herein aresubstantially isolated. By “substantially isolated” is meant that thecompound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds providedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds provided herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The present application also includes pharmaceutically acceptable saltsof the compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present application include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present application can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, non-aqueous media like ether, ethylacetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) oracetonitrile (MeCN) are preferred. Lists of suitable salts are found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2(1977). Conventional methods for preparing salt forms are described, forexample, in Handbook of Pharmaceutical Salts: Properties, Selection, andUse, Wiley-VCH, 2002.

Methods of Use

The present application further provides methods of chelating metal ionsin a sample (e.g. a cell sample or a tissue sample) or a subject,comprising contacting the sample with, or administering to the subject,a compound provided herein or (e.g., a compound of Formula I) or apharmaceutically acceptable salt thereof.

As used herein, the term “subject,” refers to any animal, includingmammals. Example subjects include, but are not limited to, mice, rats,rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans.In some embodiments, the subject is a human. In some embodiments, themethod comprises administering to the subject a therapeuticallyeffective amount of a compound provided herein (e.g., a compound of anyof Formula I), or a pharmaceutically acceptable salt thereof.

In some embodiments, the method is a method of chelating metal ions in acell or tissue sample, comprising contacting the cell sample or tissuesample with a compound provided herein, or a pharmaceutically acceptablesalt thereof. In some embodiments, the contacting forms a metal-compoundchelate.

The present application further provides a method of reducing the amountof free metal ions in a cell or tissue sample, comprising contacting thecell or tissue sample with a compound provided herein, or apharmaceutically acceptable salt thereof. In some embodiments, thecontacting forms a metal-compound chelate, thereby reducing the amountof free metal ions in the cell or tissue sample.

The present application further provides a method of chelating metalions in a subject, comprising administering to the subject atherapeutically effective amount of a compound provided herein, or apharmaceutically acceptable salt thereof. In some embodiments, the metalions are free metal ions or metal ions bound to low affinity ligands(e.g., citrate).

The present application further provides a method of reducing the amountof free metal ions in a subject, comprising administering to the subjecta compound provided herein, or a pharmaceutically acceptable saltthereof. The present application further provides a method of reducingthe amount of metal ions (e.g., free metal ions or metal ions bound tolow affinity ligands (e.g., citrate)), in the bloodstream of a subjectin need thereof. In some embodiments, the method is a method of reducingthe amount iron ions (e.g., free iron irons or iron ions bound to lowaffinity ligands (e.g., citrate)) in the bloodstream of a subject inneed thereof. In some embodiments, the subject has been determined tohave high levels of iron ions (e.g., free iron irons or iron ions boundto low affinity ligands (e.g., citrate)) in the bloodstream compared toa subject having normal levels of iron ions in the bloodstream.

The present application further provides a method of reducing iron in asubject in need thereof, comprising administering to the subject acompound provided herein, or a pharmaceutically acceptable salt thereof.In some embodiments, the method is a method of reducing iron overload ina subject in need thereof.

The present application further provides a method of reducing the amountof metal ions bound to low affinity ligands in a subject, comprisingadministering to the subject a compound provided herein, or apharmaceutically acceptable salt thereof.

The present application further provides a method of reducing the amountof metal ions in a subject, comprising:

i) diagnosing the subject as having an abnormal level of metal ions; and

ii) administering to the subject a therapeutically effective amount of acompound provided herein, or a pharmaceutically acceptable salt thereof.In some embodiments, the metal ions are free metal ions or metal ionsbound to low affinity ligands (e.g., citrate).

The present application further provides a method of reducing the amountof free metal ions in a subject, comprising:

i) diagnosing the subject as having an abnormal level of free metalions; and

ii) administering to the subject a therapeutically effective amount of acompound provided herein, or a pharmaceutically acceptable salt thereof.

The present application further provides a method of treating a diseaseassociated with an abnormal amount of metal ions in a subject. In someembodiments, the disease is associated with an abnormal amount of freemetal ions, an abnormal amount of metal ions bound to low affinityligands (e.g., citrate), or a combination thereof. In some embodiments,the method comprises administering to the subject a compound providedherein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the disease is associated with an abnormally highamount of metal ions in the subject (e.g., free metal ions, metal ionsbound to low affinity ligands, or a combination thereof) compared to asubject having normal levels of metal ions. In some embodiments, thedisease is associated with an abnormally high amount of free metal ionsin the subject, compared to a subject having normal levels of free metalions. In some embodiments, the disease is associated with an abnormallyhigh amount of metal ions bound to low affinity ligands in the subject,compared to a subject having normal levels of metal ions bound to lowaffinity ligands in the subject. In some embodiments, the disease isassociated with an abnormally high amount of a combination of free metalions and metal ions bound to low affinity ligands in the subject,compared to a subject having normal levels of free metal ions and metalions bound to low affinity ligands.

In some embodiments, the abnormal amount of metal ions in the subjectrefers to about 5% to about 100% increased concentration of metal ionsin the subject compared to the concentration of metal ions in a normalsubject, for example, about 5% to about 100%, about 5% to about 75%,about 5% to about 50%, about 5% to about 25%, about 5% to about 10%,about 10% to about 100%, about 10% to about 75%, about 10% to about 50%,about 10% to about 25%, about 25% to about 100%, about 25% to about 75%,about 25% to about 50%, about 50% to about 100%, about 50% to about 75%,or about 75% to about 100%, increased concentration of metal ions in thesubject compared to the concentration of metal ions in a normal subject.

In some embodiments, the abnormal amount of metal ions in the subjectrefers to about 2 fold to about 10 fold increased concentration of metalions in the subject compared to the concentration of metal ions in anormal subject, for example, about 2 fold to about 10 fold, about 2 foldto about 8 fold, about 2 fold to about 5 fold, about 2 fold to about 3fold, about 3 fold to about 10 fold, about 3 fold to about 8 fold, about3 fold to about 5 fold, about 5 fold to about 10 fold, about 5 fold toabout 8 fold, or about 8 fold to about 10 fold, increased concentrationof metal ions in the subject compared to the concentration of metal ionsin a normal subject.

Methods of determining the concentration of metal ions in a subject areroutine in the art and include, for example, measuring metal ions in acell sample (e.g., NIR microscopy) or tissue sample (e.g., a biopsysample by NIR spectroscopy) and/or measuring metal ions in the subjectusing an imaging technique (e.g., magnetic resonance imaging and/oroptical fluorescence imaging).

The present application further provides a method of treating a diseaseassociated with an abnormal amount of free metal ions in a subject. Insome embodiments, the method comprises administering to the subject acompound provided herein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the disease is associated with an abnormal amountof iron ions, an abnormal amount of lead ions, an abnormal amount ofcopper ions, an abnormal amount of arsenic ions, an abnormal amount ofmanganese ions, an abnormal amount of cadmium ions, an abnormal amountof nickel ions, an abnormal amount of chromium ions, an abnormal amountof gold ions, or an abnormal amount of antimony ions in the subject, orany combination thereof. In some embodiments, the disease is associatedwith an abnormal amount of iron ions, an abnormal amount of lead ions,or an abnormal amount of copper ions in the subject, or any combinationthereof.

In some embodiments, the disease is associated with an abnormal amountof iron ions, an abnormal amount of lead ions, or an abnormal amount ofcopper ions in the subject. In some embodiments, the disease isassociated with an abnormal amount of iron ions in the subject.

In some embodiments, the disease is selected from the group consistingof transfusion hemosiderosis (e.g., resulting from blood transfusions ina subject having one or more diseases selected from the group consistingof thalassemia, myelodysplastic syndrome, sickle cell anemia, andBlackfan Diamond anemia), hemochromatosis (e.g., hereditary oracquired), Wilson's disease, copper poisoning, and heavy metal poisoning(e.g., lead poisoning, mercury poisoning, cadmium poisoning, arsenicpoisoning, manganese poisoning, and the like).

In some embodiments, the compounds provided herein, or pharmaceuticallyacceptable salts thereof, are administered to the subject in atherapeutically effective amount. As used herein, the phrase“therapeutically effective amount” refers to the amount of activecompound or pharmaceutical agent that elicits the biological ormedicinal response that is being sought in a tissue, system, animal,individual or human by a researcher, veterinarian, medical doctor orother clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) inhibiting the disease; for example, inhibiting a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., arresting further development of the pathology and/orsymptomatology); and (2) ameliorating the disease; for example,ameliorating a disease, condition or disorder in an individual who isexperiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., reversing the pathology and/orsymptomatology) such as decreasing the severity of disease or reducingor alleviating one or more symptoms of the disease.

Pharmaceutical Compositions and Formulations

When employed as pharmaceuticals, the compounds and salts providedherein can be administered via various routes (e.g., intravenous,intranasal, intradermal, or oral administration) in the form ofpharmaceutical compositions. These compositions can be prepared asdescribed herein or elsewhere, and can be administered by a variety ofroutes, depending upon whether local or systemic treatment is desiredand upon the area to be treated. In some embodiments, the administrationis parenteral. Parenteral administration includes, for example,intravenous, intraarterial, subcutaneous, intraperitoneal intramuscularor injection or infusion; or intracranial administration, (e.g.,intrathecal or intraventricular, administration). Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. In some embodiments, thecompounds, salts, and pharmaceutical compositions provided herein aresuitable for parenteral administration. In some embodiments, thecompounds, salts, and pharmaceutical compositions provided herein aresuitable for intravenous administration. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable.

Also provided are pharmaceutical compositions which contain, as theactive ingredient, a compound provided herein, or a pharmaceuticallyacceptable salt thereof, in combination with one or morepharmaceutically acceptable carriers (e.g., excipients). In making thecompositions provided herein, the active ingredient is typically mixedwith an excipient, diluted by an excipient or enclosed within such acarrier in the form of, for example, a capsule, tablet, or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

Some examples of suitable excipients include, without limitation,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, and methyl cellulose. The formulations can additionally include,without limitation, lubricating agents such as talc, magnesium stearate,and mineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; flavoring agents, or combinations thereof.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual subject, the severity of thesubject's symptoms, and the like.

EXAMPLES

The following examples are offered for illustrative purposes, and arenot intended to limit the invention.

Example 1. Synthesis of ZW-EPL⁻ and DFO-ZW-EPL⁻ Nanochelators

The title compounds were prepared according to the procedure shown inScheme 1. 16 mg of ZW-EPL⁻ was dissolved in 1.5 mL water and transferredto a 10 mL round bottom flask equipped with a magnetic stirrer. About15-45 mg of DMTMM was then added to the flask and the reaction mixturewas vigorously stirred. After 5-10 min, 36 mg of DFO dissolved in 0.5 mLwater in the presence of 30-40 μL of 1 M NaOH (pH ˜7.0) was added to thereaction mixture, followed by vigorous stirring for additional 1 hour at60° C. The stoichiometry (i.e., conjugation ratio) of DFO on the EPLchain for nanochelator DFO-ZW-EPL⁻ was 1.6 as confirmed by UV-visiblespectroscopy and HPLC (see e.g., FIGS. 1A-1E). Additional DFO-ZW-EPL⁻nanochelators were also prepared at various stoichiometric ratios ofDFO:EPL, and are described below in Table 1. Absorbance spectral changesby adding DFO moieties to the DFO-ZW-EPL⁻ nanochelators were measured at200 nm (UV assay; FIG. 1F) and the absorbance changes at 430 nmreflected the number of Fe ion binding by the DFO moieties conjugated onthe ZW-EPL⁻ nanochelators (Fe assay; FIG. 1G). As shown in Table 1, thestoichiometry measured by the UV assay and Fe assay verified the numberof DFO moieties conjugated on the EPL chain.

TABLE 1 Stoichiometry (DFO:EPL) Abs at DFO Conc. UV Fe Nanochelator 200nm (μM) Assay Assay DFO-EPL-2 0.101 4.04 2.0 2.0 DFO-EPL-4 0.231 9.244.6 3.5 DFO-EPL-8 0.424 16.96 8.5 7.5

Example 2. In Vitro Iron Binding of Nanochelators

As shown in FIGS. 2A-2C, the DFO-conjugated nanochelator, DFO-ZW-EPL⁻,decreases iron concentrations in serum from iron overloaded animals inthree different assays. First, different concentrations of DFO alone(12.5 μM, 25 μM, and 50 μM), blank nanoparticles (i.e. Blank NP) andNP-DFO (50 μM as NP) were mixed with the same volume of iron solution inTBS (50 μM; pH 5.5), followed by non-heme iron analysis. Under theseconditions, the DFO-conjugated nanochelator decreased ironconcentrations in serum, as shown in FIG. 2A.

In a separate assay, sera from HFE-mutant mice, a mouse model of genetic(primary) iron overload hemochromatosis, and wild-type mice (WT; mixedstrain), were incubated with TBS (control), blank NP (100 μM) or NP-DFO(100 followed by non-heme iron analysis. Under these conditions, theDFO-conjugated nanochelator decreased the iron concentration in sera ofthe HFE-mutant mice, as shown in FIG. 2B.

Finally, sera from homozygous Belgrade (b/b) rats, a rat model oftransfusional (secondary) iron overload disorder, and healthyheterozygous control Belgrade (+/b) rats wild-type mice (mixed strain),were incubated with TBS (control), blank NP (100 μM) or NP-DFO (100 μM)before non-heme iron analysis. The DFO-conjugated nanochelator decreasediron concentration in the sera of the homozygous Belgrade (b/b) rats, asshown in FIG. 2C.

Example 3. Biodistribution of Nanochelators in CD-1 Mice

Each nanochelator was injected intravenously into 25 g CD-1 mice (10nmol; 0.2 mg/kg) 4 h prior to imaging (N=3). As shown in FIGS. 3A-3B,fluorescence images of the abdominal cavity and resected organs showlocalization of each nanochelator (DFO-ZW-EPL⁻ and ZW-EPL⁻) in thekidney and bladder.

Example 4. Pharmacokinetics of Nanochelators

An overview of the pharmacokinetics of nanochelators ZW-EPL⁻ andDFO-ZW-EPL⁻ are shown below in Table 2, and in FIGS. 4A-4C.

TABLE 2 DFO-ZW-EPL⁻ ZW-EPL⁻ MW (g/mol) ~8,700 ~7,600 D (μg) 87 76 K(/min) 0.0140 0.0049 T_(1/2) α (min) 3.44 ± 0.26 3.98 ± 0.25 T_(1/2) β(min) 49.46 ± 29.93 140 ± 86  Urinary excretion (% ID) 45.44 64.46 AUC(% ID min) 224.2 1677 Plasma clearance (mL/min) 0.446 0.060 volume ofdistribution (mL) 31.83 12.13

Example 5. In Vivo Iron Chelation Effect of Nanochelators in DietaryIron Overload Rats

Rats (female) were treated with iron loading diet (10,000 ppm) for 2weeks prior to the experiment. DFO (30 mg/kg) and DFO-ZW-EPL⁻ (0.3 mg/kgas DFO and 1 μmol/kg as NP) were filtered and injected (i.v.) throughtail vein (1 mL/kg). Blood was collected at 5 min, 1 h, 2 h, 3 h and 4 hpost-administration. After 4 h, rats were euthanized and tissues werecollected. Serum iron and liver non-heme iron levels were quantified bynon-heme iron analysis. As shown in FIGS. 5A-5B, administration ofcompound DFO-ZW-EPL⁻ increased in vivo iron chelator efficacy in serumby approximately 100 fold compared to administration of free DFO (FIG.5A) and reduced the measured levels of liver non-heme iron compared toadministration of DFO alone (FIG. 5B).

Example 6. In Vivo Iron Chelation Effect of Nanochelators in Mice withGenetic Iron Overload

Nanochelators display improved iron chelation effect in a mouse model ofiron overload hemochromatosis. HFE mutant mice (male and female), amouse model of genetic iron overload, were intravenously injectedthrough the tail vein with saline (no DFO), DFO alone (0.3 mg/kg, 30mg/kg and 300 mg/kg) or DFO-ZW-EPL⁻ (i.e., NP-DFO; 0.3 mg/kg as DFO and1 μmol/kg as NP). Blood was taken at 5 min, 1 h, 2 h, 3 h, and 4 h.After 4 hours, mice were euthanized and tissues were collected. Serumiron and liver non-heme iron levels were quantified by non-heme ironanalysis, as shown in FIGS. 6A-6B.

In a separate assay, HFE-mutant mice (n=2/group for EPL; n=3 forDFO-EPL-2 and DFO-EPL-4; n=1 for DFO-EPL-8) were administered byintravenous (i.v.) injection with EPL (1 μmol/kg) or DFO-EPL (1 μmol/kgas of EPL). Blood was collected at designated time pointspost-administration. Serum iron levels were quantified by colorimetricanalysis and the data are shown in FIG. 6C (mean±SEM). Serum iron levelswere significantly decreased within 4 h in DFO-EPL-8 treated mice(DFO:NP stoichiometry=8:1), but not in mice treated with blank NPs.

In a separate assay, HFE-deficient mice were administered bysubcutaneous (s.c.) injection with saline, blank EPL (1 μmol/kg),DFO-EPL-2 and DFO-EPL-4 (1 μmol/kg as of EPL) (n=2/group). Blood wastaken before dosing at designated time points for 5 days and normalizedto values from the saline-treated control group. Serum iron levels werequantified by colorimetric analysis, and the data are shown in FIG. 6D(mean±SEM). Again, DFO-NPs decreased serum iron in a dose-dependentmanner whereas blank NPs did not, indicating that the DFO-NPs candecrease iron burden in iron overload disorders.

Example 7. Therapeutic Efficacy of Nanochelators in Cardiac HypertrophyAssociated with Iron Overload

It will be determined if the nanochelators (e.g., DFO-ZW-EPL⁻) correctcardiac hypertrophy in HFE-knockout mice after repeated/sub-chronicdosing and by dose-response relationships). Measurements may include:heart/body weight ratio, fluorescent microscopy for cardiac hypertrophy,western blot analysis, hypertrophic gene expression, levels of oxidativestress markers, and serum cardiac troponin levels (see e.g., FIGS.7A-7B).

Example 8. Efficacy of Nanochelators in Impaired Lipid MetabolismAssociated with Iron Overload

It will be determined if the nanochelators (e.g., DFO-ZW-EPL⁻) restoredyslipidemia in Belgrade rats after repeated/sub-chronic dosing and bydose-response relationships). Measurements may include: blood glucoselevels, cholesterol levels, triglyceride levels, free fatty acid levels,lipoprotein lipase activity, and cardiac hypertrophy (see e.g., FIGS.8A-8G).

Example 9. In Vivo Iron Chelation Effect of Nanochelators in Mice withTransfusional Iron Overload

The efficacy of DFO-NPs was evaluated in mice that were treated withiron-dextran, a model of transfusional iron overload (see e.g, Imranul-haq et al, ACS Nano, 2013, 7:10704-10716; and Liu et al, ACS. Appl.Mater. Interfaces, 2016, 8:25788-25797). CD-1 mice were pretreated withiron dextran (100 mg/kg) by intraperitoneal injection and thenintravenously injected with DFO-NPs, blank NPs (1 μmol as NP/kg) orsaline 4 h post-iron dextran treatment. Urine was collected for 4 hafter NP administration to determine renal excretion of iron. Ironrecovery in urine was significantly increased in DFO-NP administeredmice compared with saline- or blank NP-injected mice as shown in FIG. 9,confirming that the DFO-NPs collected excessive iron from circulationand excreted via urine. The pharmacokinetics of DFO-NPs in CD-1 micedemonstrated that over 40% of injected dose of DFO-NPs was excreted intourine within 4 h after intravenous injection with negligibledistribution in other organs.

In a separate assay, CD-1 mice were administered by subcutaneous (s.c.)injection with native DFO alone (8 μmol/kg), blank EPL (1 μmol/kg),DFO-EPL-8 (1 μmol/kg as EPL) daily for 5 days. Another CD-1 mouse wasinjected a single dose of DFO-EPL (1 μmol/kg). One day after last dose,the mice were euthanized, followed by NIR fluorescence. The amount ofDFO-NPs found in the kidney was not different between single-dose andmulti-dose studies, as shown in FIG. 10. Without being bound by anytheory, this data is believed to show that the NPs are rapidly excretedwithout significant accumulation in the kidney.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of chelating metal ions in a subject,comprising administering to the subject a therapeutically effectiveamount of a compound of Formula I:AB   I or a pharmaceutically acceptable salt thereof, wherein: A isselected from the group consisting of formulas A-1 and A-2:

wherein:

indicates the bond between A and B; X is selected from the groupconsisting of a bond, C, CH₂, NH, O and S; each R^(A) is anindependently selected anionic group selected from the group consistingof oxide, carbonate, carboxylate, phosphate, sulfide, sulfinate, andsulfonate; each R^(C) is an independently selected cationic groupselected from the group consisting of ammonium, C₁₋₆ alkylammonium,di(C₁₋₆ alkyl)ammonium, and tri(C₁₋₆ alkyl)ammonium; and L¹, L², and L³are each an independently selected C₁₋₆ alkylene group; B is abiocompatible polymer substituted by one or more C groups and one ormore -D-E groups; each C is independently selected from the groupconsisting of H and an anionic group; each D is an independentlyselected linking group; and each E is an independently selected metalchelating group.
 2. The method of claim 1, wherein the method comprisesreducing the amount of free metal ions in the subject.
 3. A method oftreating a disease associated with an abnormal amount of free metal ionsin a subject, comprising administering to a subject determined to havean abnormal level of free metal ions a compound of Formula I:AB   I or a pharmaceutically acceptable salt thereof, wherein: A isselected from the group consisting of formulas A-1 and A-2:

wherein:

indicates the bond between A and B; X is selected from the groupconsisting of a bond, C, CH₂, NH, O and S; each R^(A) is anindependently selected anionic group selected from the group consistingof oxide, carbonate, carboxylate, phosphate, sulfide, sulfinate, andsulfonate; each R^(C) is an independently selected cationic groupselected from the group consisting of ammonium, C₁₋₆ alkylammonium,di(C₁₋₆ alkyl)ammonium, and tri(C₁₋₆ alkyl)ammonium; and L¹, L², and L³are each an independently selected C₁₋₆ alkylene group; B is abiocompatible polymer substituted by one or more C groups and one ormore -D-E groups; each C is independently selected from the groupconsisting of H and an anionic group; each D is an independentlyselected linking group; and each E is an independently selected metalchelating group.
 4. The method of claim 3, wherein the disease isassociated with an abnormal amount of iron ions, an abnormal amount oflead ions, or an abnormal amount of copper ions in the subject, or anycombination thereof.
 5. The method of claim 3, wherein the disease isassociated with an abnormal amount of iron ions in the subject.
 6. Themethod of claim 3, wherein the disease is selected from the groupconsisting of transfusion hemosiderosis, hemochromatosis, Wilson'sdisease, copper poisoning, and heavy metal poisoning.
 7. The method ofclaim 3, wherein the method comprises reducing the amount of free metalions or metal ions bound to low affinity ligands, in the bloodstream ofthe subject.
 8. The method of claim 3, wherein A is:


9. The method of claim 3, wherein B is selected from the groupconsisting of polylysine, polylactic acid, poly(lactic-co-glycolicacid), polyaspartic acid, polyglutamic acid, and polyglutamicacid-poly(ethylene glycol) copolymer, each of which is substituted byone or more C groups and one or more -D-E groups.
 10. The method ofclaim 3, wherein each C is independently selected from the groupconsisting of hydrogen and an anionic group comprising one or morealkylene groups, one or more carbonyl groups, or one or more carboxylgroups, or any combination thereof.
 11. The method of claim 3, wherein Dis a linking group comprising one or more alkylene groups, one or morecarbonyl groups, or one or more carboxyl groups, or any combinationthereof.
 12. The method of claim 3, wherein E is selected from the groupconsisting of an iron chelating group, a lead chelating group, a copperchelating group, an arsenic chelating group, a mercury chelating group,and a manganese chelating group.
 13. The method of claim 3, wherein: Bis selected from the group consisting of a biocompatible polypeptide anda biocompatible polyester, each of which is substituted by one or more Cgroups and one or more -D-E groups; C is an anionic group of thefollowing formula:

wherein:

indicates the bond between C and B; p is an integer from 1 to 10; D is alinking group of the following formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and q is an integer from 1 to 10;and E is a metal chelating group.
 14. The method of claim 13, wherein Eis selected from the group consisting of an iron chelating group, a leadchelating group, and a copper chelating group.
 15. The method of claim3, wherein E is an iron chelating group.
 16. The method of claim 3,wherein E is selected from the group consisting of dimercaptosuccinicacid, dimercaprol, ethylenediaminetetraacetic acid, p-aminosalicyclicacid, D-penicillamine, deferoxamine, deferiprone, and deferasirox. 17.The method of claim 3, wherein E is deferoxamine.
 18. The method ofclaim 3, wherein X is a bond or CH₂.
 19. The method of claim 3, whereineach R^(C) is an independently selected tri(C₁₋₆ alkyl)ammonium group.20. The method of claim 3, wherein each R^(A) is sulfonate.
 21. Themethod of claim 3, wherein B is polylysine substituted by one or more Cgroups and one or more -D-E groups.
 22. The method of claim 3, wherein Bis:

wherein:

indicates the bond between B and A; and n is an integer from 5 to 30;and m is an integer from 1 to
 10. 23. The method of claim 3, wherein Cis an anionic group of the following formula:

wherein:

indicates the bond between C and B; and p is an integer from 1 to 10.24. The method of claim 3, wherein: D is a linking group of thefollowing formula:

wherein:

indicates the bond between D and B;

indicates the bond between D and E; and q is an integer from 1 to 10.25. The method of claim 20, wherein E is selected from the groupconsisting of dimercaptosuccinic acid, dimercaprol,ethylenediaminetetraacetic acid, p-aminosalicyclic acid,D-penicillamine, deferoxamine, deferiprone, and deferasirox.
 26. Themethod of claim 3, wherein the compound of Formula I, or apharmaceutically acceptable salt thereof, is administered to the subjectin the form of a pharmaceutical composition comprising the compound ofFormula I, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.